The cyclin/CDK inhibitor p21 Cip1Waf1 participates in a number of protein-protein interactions that bear on cell-cycle control, apoptosis and transcription. In keratinocytes, p21 plays a dual biological function: (1) it restricts the size of keratinocyte stem cell populations and promotes the irreversible commitment to differentiation; (2) it is part of a negative regulatory mechanism that needs to be inactivated for the late stages of differentiation. In our further work on this to principal investigator, we will test the hypothesis that p21 is involved in transcription control of specific genes linked to keratinocyte growth/differentiation control. This may occur, in part, independently from the cell cycle and in a cell-type specific manner. Our initial approach will involve global analysis of gene expression in p21+/+ versus p21-/- keratinocytes, under growing versus differentiating conditions, and in cells expressing the full length p21 protein versus the p21 amino-terminus domain (which suppresses growth without affecting differentiation). This will be followed by direct functional studies to assess whether p21 restricts the keratinocyte stem cell potential and promotes the irreversible commitment to differentiation through modulation of specific cell regulatory genes. For this, individual genes that are concordantly modulated by increased p21 expression and differentiation in p21 +/+ but not p21-/- cells will be tested for their effects by a combination of in vitro and in vivo approaches. In our preliminary work, we have found that increased p21 expression suppresses the late stages of differentiation through activation of kinases of the MAPK family. Thus, we will test the hypothesis that the inhibitory effects of p21 on differentiation are mediated by modulation of genes with MAPK activation potential, independently of the cell cycle and independently and/or in opposition to differentiation.